Advertisement

Clinical Oral Investigations

, Volume 18, Issue 2, pp 659–669 | Cite as

pH gradient and distribution of streptococci, lactobacilli, prevotellae, and fusobacteria in carious dentine

  • Nima KianoushEmail author
  • Ky-Anh T. Nguyen
  • Gina V. Browne
  • Mary Simonian
  • Neil Hunter
Original Article

Abstract

Objectives

Caries process comprises acidogenic and aciduric bacteria that are responsible for lowering the pH and subsequent destruction of hydroxyapatite matrix in enamel and dentine. The aim of this study was to identify the correlation between the pH gradient of a carious lesion and proportion and distribution of four bacterial genera; lactobacilli, streptococci, prevotellae, and fusobacteria with regard to total load of bacteria.

Materials and methods

A total of 25 teeth with extensive dentinal caries were sampled in sequential layers. Using quantitative real-time PCR of 16S rRNA gene, we quantified the total load of bacteria as well as the proportion of the above-mentioned genera following pH measurement of each sample with a fine microelectrode.

Results

We demonstrated the presence of a pH gradient across the lesion with a strong association between the quantity of lactobacilli and the lowest pH range (pH 4.5–5.0; p = 0.003). Streptococci had a tendency to occupy the most superficial aspect of the carious lesion but showed no correlation to any pH value. Prevotellae showed clear preference for the pH range 5.5–6.0 (p = 0.042). The total representation of these four genera did not reach more than one quarter of the total bacterial load in most carious samples.

Conclusion

We revealed differential colonization behavior of bacteria with respect to pH gradient and a lower than expected abundance of lactobacilli and streptococci in established carious lesions. The data indicate the numerical importance of relatively unexplored taxa within the lesion of dentinal caries.

Clinical relevance

The gradient nature of pH in the lesion as well as colonization difference of examined bacterial taxa with reference to pH provides a new insight in regard to conservative caries management.

Keywords

Caries Real-time PCR Gram-positive bacteria Gram-negative bacteria pH gradient 16S rRNA 

Notes

Acknowledgments

The authors acknowledge funding from NIH/NIDCR (grant no. R01 DEO15272-07), Australian National Health and Medical Research Council (NHMRC grant no. 512524.3), Australian Dental Research Foundation (grant no. 91–2011]), and New South Wales Dental Board (grant no. 2011). We thank Dr Derek Harty and Dr Mangala Nadkarni (Institute of Dental Research, Westmead, NSW, Australia) for provision of some DNA materials. We also thank Mr. Mitchell Brown and Mr. Terry Flood (Center for Infectious Disease and Microbiology, Institute for Clinical Pathology and Medical Research, Westmead, Australia) who provided us with valuable assistance with culture and magnetic DNA extraction of some bacteria.

Conflict of interest

The authors declare no potential conflicts of interest in regard to authorship and/or publication of this article.

Supplementary material

784_2013_1009_MOESM1_ESM.pdf (340 kb)
ESM 1 (PDF 340 kb)
784_2013_1009_MOESM2_ESM.xlsx (33 kb)
ESM Table 1 (XLSX 33 kb)

References

  1. 1.
    Featherstone JD (2004) The continuum of dental caries--evidence for a dynamic disease process. J Dent Res 83 Spec No C: C39-42Google Scholar
  2. 2.
    van Houte J (1994) Role of microorganisms in caries etiology. J Dent Res 73(3):672–681PubMedGoogle Scholar
  3. 3.
    Chhour KL, Nadkarni MA, Byun R, Martin FE, Jacques NA, Hunter N (2005) Molecular analysis of microbial diversity in advanced caries. J Clin Microbiol 43(2):843–849. doi: 10.1128/JCM.43.2.843-849.2005 PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Nadkarni MA, Caldon CE, Chhour KL, Fisher IP, Martin FE, Jacques NA, Hunter N (2004) Carious dentine provides a habitat for a complex array of novel Prevotella-like bacteria. J Clin Microbiol 42(11):5238–5244. doi: 10.1128/JCM.42.11.5238-5244.2004 PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Takahashi N, Saito K, Schachtele CF, Yamada T (1997) Acid tolerance and acid-neutralizing activity of Porphyromonas gingivalis, Prevotella intermedia, and Fusobacterium nucleatum. Oral Microbiol Immunol 12(6):323–328PubMedCrossRefGoogle Scholar
  6. 6.
    Marsh PD (1994) Microbial ecology of dental plaque and its significance in health and disease. Adv Dent Res 8(2):263–271PubMedGoogle Scholar
  7. 7.
    Takahashi N, Nyvad B (2011) The role of bacteria in the caries process: ecological perspectives. J Dent Res 90(3):294–303. doi: 10.1177/0022034510379602 PubMedCrossRefGoogle Scholar
  8. 8.
    Stephan RM (1940) Two factors of possible importance in relation to the etiology and treatment of dental caries and other dental diseases. Science 92(2399):578–579. doi: 10.1126/science.92.2399.578 PubMedCrossRefGoogle Scholar
  9. 9.
    Featherstone JD, Rodgers BE (1981) Effect of acetic, lactic, and other organic acids on the formation of artificial carious lesions. Caries Res 15(5):377–385PubMedCrossRefGoogle Scholar
  10. 10.
    Macgregor AB (1961) The position and extent of acid in the carious process. Archives of oral biology 4:86–91PubMedCrossRefGoogle Scholar
  11. 11.
    Van Houte J, Sansone C, Joshipura K, Kent R (1991) Mutans streptococci and non-mutans streptococci acidogenic at low pH and in vitro acidogenic potential of dental plaque in two different areas of the human dentition. J Dent Res 70(12):1503–1507PubMedCrossRefGoogle Scholar
  12. 12.
    Takahashi N, Yamada T (1999) Acid-induced acid tolerance and acidogenicity of non-mutans streptococci. Oral Microbiol Immunol 14(1):43–48PubMedCrossRefGoogle Scholar
  13. 13.
    Iwami Y, Abbe K, Takahashi-Abbe S, Yamada T (1992) Acid production by streptococci growing at low pH in a chemostat under anaerobic conditions. Oral Microbiol Immunol 7(5):304–308PubMedCrossRefGoogle Scholar
  14. 14.
    Badet MC, Richard B, Dorignac G (2001) An in vitro study of the pH-lowering potential of salivary lactobacilli associated with dental caries. J Appl Microbiol 90(6):1015–1018PubMedCrossRefGoogle Scholar
  15. 15.
    McLean JS, Fansler SJ, Majors PD, McAteer K, Allen LZ, Shirtliff ME, Lux R, Shi W (2012) Identifying low pH active and lactate-utilizing taxa within oral microbiome communities from healthy children using stable isotope probing techniques. PLoS One 7(3):e32219. doi: 10.1371/journal.pone.0032219 PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Hojo S, Komatsu M, Okuda R, Takahashi N, Yamada T (1994) Acid profiles and pH of carious dentin in active and arrested lesions. J Dent Res 73(12):1853–1857PubMedGoogle Scholar
  17. 17.
    Byun R, Nadkarni MA, Chhour KL, Martin FE, Jacques NA, Hunter N (2004) Quantitative analysis of diverse Lactobacillus species present in advanced dental caries. J Clin Microbiol 42(7):3128–3136. doi: 10.1128/JCM.42.7.3128-3136.2004 PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Martin FE, Nadkarni MA, Jacques NA, Hunter N (2002) Quantitative microbiological study of human carious dentine by culture and real-time PCR: association of anaerobes with histopathological changes in chronic pulpitis. J Clin Microbiol 40(5):1698–1704PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23(21):2947–2948. doi: 10.1093/bioinformatics/btm404 PubMedCrossRefGoogle Scholar
  20. 20.
    Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2 PubMedGoogle Scholar
  21. 21.
    Nadkarni MA, Martin FE, Jacques NA, Hunter N (2002) Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set. Microbiology 148(Pt 1):257–266PubMedGoogle Scholar
  22. 22.
    Smith CJ, Osborn AM (2009) Advantages and limitations of quantitative PCR (Q-PCR)-based approaches in microbial ecology. FEMS Microbiol Ecol 67(1):6–20. doi: 10.1111/j.1574-6941.2008.00629.x PubMedCrossRefGoogle Scholar
  23. 23.
    Nadkarni MA, Martin FE, Hunter N, Jacques NA (2009) Methods for optimizing DNA extraction before quantifying oral bacterial numbers by real-time PCR. FEMS Microbiol Lett 296(1):45–51. doi: 10.1111/j.1574-6968.2009.01629.x PubMedCrossRefGoogle Scholar
  24. 24.
    Lee ZM, Bussema C, 3rd, Schmidt TM (2009) rrnDB: documenting the number of rRNA and tRNA genes in bacteria and archaea. Nucleic Acids Res 37 (Database issue):D489-493. doi:  10.1093/nar/gkn689
  25. 25.
    Holm S (1979) A simple sequentially rejective multiple test procedure. Scand J Statist 6:65–70Google Scholar
  26. 26.
    Schachtele CF, Jensen ME (1982) Comparison of methods for monitoring changes in the pH of human dental plaque. J Dent Res 61(10):1117–1125PubMedCrossRefGoogle Scholar
  27. 27.
    Kleinberg I, Jenkins GN, Chatterjee R, Wijeyeweera L (1982) The antimony pH electrode and its role in the assessment and interpretation of dental plaque pH. J Dent Res 61(10):1139–1147PubMedCrossRefGoogle Scholar
  28. 28.
    Stephen RM (1940) Changes in hydrogen ion concentration on tooth surfaces and in carious lesions. J Am Dent Assoc 27:718–723Google Scholar
  29. 29.
    Bentley KD, Haldi J, Law ML, Ramsey DA, Wynn W (1956) Dental caries in relation to pH on tooth surfaces. I. pH and lactate concentration in relation to the extent of the lesions in rats' teeth. J Nutr 60(3):427–435PubMedGoogle Scholar
  30. 30.
    Igarashi K, Kamiyama K, Yamada T (1981) Measurement of pH in human dental plaque in vivo with an ion-sensitive transistor electrode. Archives of oral biology 26(3):203–207PubMedCrossRefGoogle Scholar
  31. 31.
    Hassell TM (1971) Construction of micro-antimony electrodes for use in radio telemetry of plaque pH. Helvetica odontologica acta 15(1):50–51PubMedGoogle Scholar
  32. 32.
    Tahmassebi JF, Duggal MS (1997) The effect of different methods of drinking on the pH of dental plaque in vivo. Intern J Paediatr Dent Br Paedodontic Soc Intern Assoc Dent Children 7(4):249–254Google Scholar
  33. 33.
    Roos EH, Donly KJ (2002) In vivo dental plaque pH variation with regular and diet soft drinks. Pediatr Dent 24(4):350–353PubMedGoogle Scholar
  34. 34.
    Jawale BA, Bendgude V, Mahuli AV, Dave B, Kulkarni H, Mittal S (2012) Dental plaque pH variation with regular soft drink, diet soft drink, and high energy drink: an in vivo study. J Contemp Dent Pract 13(2):201–204PubMedGoogle Scholar
  35. 35.
    Dirksen TR, Little MF, Bibby BG (1963) The pH of carious cavities-II. The pH at different depths in isolated cavities. Archives of oral biology 8:91–97PubMedCrossRefGoogle Scholar
  36. 36.
    Hojo S, Takahashi N, Yamada T (1991) Acid profile in carious dentin. J Dent Res 70(3):182–186PubMedCrossRefGoogle Scholar
  37. 37.
    Hopkins MJ, Englyst HN, Macfarlane S, Furrie E, Macfarlane GT, McBain AJ (2003) Degradation of cross-linked and non-cross-linked arabinoxylans by the intestinal microbiota in children. Appl Environ Microbiol 69(11):6354–6360PubMedCentralPubMedCrossRefGoogle Scholar
  38. 38.
    Jansen HJ, van der Hoeven JS (1997) Protein degradation by Prevotella intermedia and Actinomyces meyeri supports the growth of nonprotein-cleaving oral bacteria in serum. J Clin Periodontol 24(5):346–353PubMedCrossRefGoogle Scholar
  39. 39.
    Takahashi N (2003) Acid-neutralizing activity during amino acid fermentation by Porphyromonas gingivalis, Prevotella intermedia, and Fusobacterium nucleatum. Oral Microbiol Immunol 18(2):109–113PubMedCrossRefGoogle Scholar
  40. 40.
    Bradshaw DJ, Marsh PD (1998) Analysis of pH-driven disruption of oral microbial communities in vitro. Caries Res 32(6):456–462PubMedCrossRefGoogle Scholar
  41. 41.
    Periasamy S, Kolenbrander PE (2010) Central role of the early colonizer Veillonella sp. in establishing multispecies biofilm communities with initial, middle, and late colonizers of enamel. J Bacteriol 192(12):2965–2972. doi: 10.1128/JB.01631-09 PubMedCentralPubMedCrossRefGoogle Scholar
  42. 42.
    Chalmers NI, Palmer RJ Jr, Cisar JO, Kolenbrander PE (2008) Characterization of a Streptococcus sp.–Veillonella sp. community micromanipulated from dental plaque. J Bacteriol 190(24):8145–8154. doi: 10.1128/JB.00983-08 PubMedCentralPubMedCrossRefGoogle Scholar
  43. 43.
    Horiuchi M, Washio J, Mayanagi H, Takahashi N (2009) Transient acid-impairment of growth ability of oral Streptococcus, Actinomyces, and Lactobacillus: a possible ecological determinant in dental plaque. Oral Microbiol Immunol 24(4):319–324. doi: 10.1111/j.1399-302X.2009.00517.x PubMedCrossRefGoogle Scholar
  44. 44.
    Berkovitz BKB, Holland GR, Moxham BJ (2002) Oral anatomy, histology and embryology. 3rd edn. MosbyGoogle Scholar
  45. 45.
    Vanuspong W, Eisenburger M, Addy M (2002) Cervical tooth wear and sensitivity: erosion, softening, and rehardening of dentine: effects of pH, time, and ultrasonication. J Clin Periodontol 29(4):351–357PubMedCrossRefGoogle Scholar
  46. 46.
    Marsh PD (2003) Are dental diseases examples of ecological catastrophes? Microbiology 149(Pt 2):279–294PubMedCrossRefGoogle Scholar
  47. 47.
    Bradshaw DJ, Marsh PD, Allison C, Schilling KM (1996) Effect of oxygen, inoculum composition, and flow rate on development of mixed-culture oral biofilms. Microbiology 142(Pt 3):623–629PubMedCrossRefGoogle Scholar
  48. 48.
    Munson MA, Banerjee A, Watson TF, Wade WG (2004) Molecular analysis of the microflora associated with dental caries. J Clin Microbiol 42(7):3023–3029. doi: 10.1128/JCM.42.7.3023-3029.2004 PubMedCentralPubMedCrossRefGoogle Scholar
  49. 49.
    van Houte J, Lopman J, Kent R (1994) The predominant cultivable flora of sound and carious human root surfaces. J Dent Res 73(11):1727–1734PubMedGoogle Scholar
  50. 50.
    Sansone C, Van Houte J, Joshipura K, Kent R, Margolis HC (1993) The association of mutans streptococci and non-mutans streptococci capable of acidogenesis at a low pH with dental caries on enamel and root surfaces. J Dent Res 72(2):508–516PubMedCrossRefGoogle Scholar
  51. 51.
    Loesche WJ (1986) Role of Streptococcus mutans in human dental decay. Microbiol Rev 50(4):353–380PubMedCentralPubMedGoogle Scholar
  52. 52.
    Gross EL, Beall CJ, Kutsch SR, Firestone ND, Leys EJ, Griffen AL (2012) Beyond Streptococcus mutans: dental caries onset linked to multiple species by 16S rRNA community analysis. PLoS One 7(10):e47722. doi: 10.1371/journal.pone.0047722 PubMedCentralPubMedCrossRefGoogle Scholar
  53. 53.
    Aas JA, Griffen AL, Dardis SR, Lee AM, Olsen I, Dewhirst FE, Leys EJ, Paster BJ (2008) Bacteria of dental caries in primary and permanent teeth in children and young adults. J Clin Microbiol 46(4):1407–1417. doi: 10.1128/JCM.01410-07 PubMedCentralPubMedCrossRefGoogle Scholar
  54. 54.
    Gross EL, Leys EJ, Gasparovich SR, Firestone ND, Schwartzbaum JA, Janies DA, Asnani K, Griffen AL (2010) Bacterial 16S sequence analysis of severe caries in young permanent teeth. J Clin Microbiol 48(11):4121–4128. doi: 10.1128/JCM.01232-10 PubMedCentralPubMedCrossRefGoogle Scholar
  55. 55.
    Jenkinson HF (2011) Beyond the oral microbiome. Environ Microbiol 13(12):3077–3087. doi: 10.1111/j.1462-2920.2011.02573.x PubMedCrossRefGoogle Scholar
  56. 56.
    Klein D (2002) Quantification using real-time PCR technology: applications and limitations. Trends Mol Med 8(6):257–260PubMedCrossRefGoogle Scholar
  57. 57.
    Smith CJ, Nedwell DB, Dong LF, Osborn AM (2006) Evaluation of quantitative polymerase chain reaction-based approaches for determining gene copy and gene transcript numbers in environmental samples. Environ Microbiol 8(5):804–815. doi: 10.1111/j.1462-2920.2005.00963.x PubMedCrossRefGoogle Scholar
  58. 58.
    Love JL, Scholes P, Gilpin B, Savill M, Lin S, Samuel L (2006) Evaluation of uncertainty in quantitative real-time PCR. J Microbiol Methods 67(2):349–356. doi: 10.1016/j.mimet.2006.04.005 PubMedCrossRefGoogle Scholar
  59. 59.
    Frencken JE, Leal SC, Navarro MF (2012) Twenty-five-year atraumatic restorative treatment (ART) approach: a comprehensive overview. Clinical oral investigations 16(5):1337–1346. doi: 10.1007/s00784-012-0783-4 PubMedCentralPubMedCrossRefGoogle Scholar
  60. 60.
    Topaloglu-Ak A, Eden E, Frencken JE, Oncag O (2009) Two-year survival rate of class II composite resin restorations prepared by ART with and without a chemomechanical caries removal gel in primary molars. Clinical oral investigations 13(3):325–332. doi: 10.1007/s00784-008-0241-5 PubMedCentralPubMedCrossRefGoogle Scholar
  61. 61.
    Eden E, Topaloglu-Ak A, Frencken JE, van't Hof M (2006) Survival of self-etch adhesive Class II composite restorations using ART and conventional cavity preparations in primary molars. Am J Dent 19(6):359–363PubMedGoogle Scholar
  62. 62.
    Louw AJ, Sarvan I, Chikte UM, Honkala E (2002) One-year evaluation of atraumatic restorative treatment and minimum intervention techniques on primary teeth. SADJ: J S Afr Dental Assoc = tydskrif van die Suid-Afrikaanse Tandheelkundige Vereniging 57(9):366–371Google Scholar
  63. 63.
    Cefaly DF, Barata TJ, Bresciani E, Fagundes TC, Lauris JR, Navarro MF (2007) Clinical evaluation of multiple-surface ART restorations: 12-month follow-up. J Dent Child (Chic) 74(3):203–208Google Scholar
  64. 64.
    Faccin ES, Ferreira SH, Kramer PF, Ardenghi TM, Feldens CA (2009) Clinical performance of ART restorations in primary teeth: a survival analysis. J Clin Pediatr Dent 33(4):295–298PubMedGoogle Scholar
  65. 65.
    Taifour D, Frencken JE, Beiruti N, van 't Hof MA, Truin GJ (2002) Effectiveness of glass-ionomer (ART) and amalgam restorations in the deciduous dentition: results after 3 years. Caries Res 36 (6):437–444. doi:66531Google Scholar
  66. 66.
    Beiruti N, Frencken JE, van't Hof MA, Taifour D, van Palenstein Helderman WH (2006) Caries-preventive effect of a one-time application of composite resin and glass ionomer sealants after 5 years. Caries Res 40(1):52–59. doi: 10.1159/000088907 PubMedCrossRefGoogle Scholar
  67. 67.
    Zanata RL, Fagundes TC, Freitas MC, Lauris JR, Navarro MF (2011) Ten-year survival of ART restorations in permanent posterior teeth. Clin Oral Inv 15(2):265–271. doi: 10.1007/s00784-009-0378-x CrossRefGoogle Scholar
  68. 68.
    Prentice LH, Tyas M, Burrow MF (2006) The effect of oxalic acid incorporation on the setting time and strength of glass-ionomer cement. Acta biomaterialia 2(1):109–112. doi: 10.1016/j.actbio.2005.08.007 PubMedCrossRefGoogle Scholar
  69. 69.
    McInnes-Ledoux PM, Weinberg R, Grogono A (1989) Bonding glass-ionomer cements to chemomechanically-prepared dentin. Dent Mater Off Publ Acad Dent Mater 5(3):189–193Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Nima Kianoush
    • 1
    • 2
    • 3
    Email author
  • Ky-Anh T. Nguyen
    • 1
    • 2
  • Gina V. Browne
    • 1
  • Mary Simonian
    • 1
  • Neil Hunter
    • 1
    • 2
  1. 1.Institute of Dental Research, Westmead Center for Oral Health and Westmead Millennium InstituteSydneyAustralia
  2. 2.Department of Oral Biology, Faculty of DentistryUniversity of SydneySydneyAustralia
  3. 3.Department of Oral Biology, Institute of Dental ResearchLevel 2, Westmead Center for Oral Health C24WentworthvilleAustralia

Personalised recommendations